While the immune response against viral pathogens is strong, antitumor immunity is weak. This is largely due to the ability of viral elements to activate innate immune cells through toll like receptors (TLRs). However, tumors, which are derived from host tissues, are incapable of activating a strong innate immune response. Plasmacytoid dendritic cells (pDCs) are central to the potent innate immune response against viruses and lead directly to the activation of other immune cells, including myeloid dendritic cells and natural killer cells which then lead to a potent adaptive T-cell immune response. The innate immune response is important in not only triggering strong T-cell priming, but also in inducing inflammation at the site of pathogen which leads to migration of primed T-cells to the infected site. It is our goal to utilize these principles to generate a strong antitumor immune response. The overall hypothesis is that activation of plasmacytoid dendritic cells using TLR agonists will result in an inflammatory cascade that will lead to both improved T-cell priming as well as enhanced migration to and function at the tumor site. Four integrated projects and three cores are proposed to evaluate the role of plasmacytoid dendritic cells in antitumor immunity and identify methods to improve vaccine strategies. These projects represent a systematic evaluation of this issue from laboratory to clinical endpoints. This includes a mechanistic evaluation of plasmacytoid dendritic cell interactions, studies of pDCs in murine and human model systems, a clinical vaccine trial utilizing TLR agonists to activate pDCs in vivo, and the integration of these studies. Project 1 will focus on evaluating the molecular interactions between human pDCs, natural killer (NK) cells, and B lymphocytes, in the presence and absence of activation by TLR agonists. In Project 2, further characterization of pDC interactions will be performed in a murine tumor model. In Project 3, we will examine endogenous pDC in human tumor samples. Our preliminary evidence suggests that pDC in the tumor site are not functional due to lack of activation stimuli. In Project 4, we will attempt to provide these pDC activation signals in patients by the administration of TLR agonists at the vaccine site to induce the activation and proliferation of tumor-specific T-cells as well as systemically to induce inflammation in metastatic lesions in order to enhance T-cell migration to and function at the tumor site. The resources provided by the Administrative Core, the Biostatistical and Data Management Core, and the Immune Monitoring Core are crucial to this effort, and will provide the resources and structure for the seamless integration and evaluation of samples and analyses of data. The goal of this application is to apply the basic principles gleaned from analysis of successful antiviral immune responses to the development of improved antitumor immune responses, and these studies may produce findings that can be applicable to other cancers.